Substrate targeting mechanism of a DesCEND pathway

摘要 该补助金R 01 GM 135592的补充申请建议采购液相色谱仪， 蛋白质纯化系统，以支持研究目标，重点是调查底物靶向 SCF泛素连接酶F-box亚基FBXO 31在新型DesCEND途径中的作用机制。通过 在系统水平和分子水平上识别和表征FBXO 31的底物，我们 提出阐明FBXO 31-降解决定子对的互补结构决定因素， 底物特异性和决定蛋白质识别和命运。为了实现我们的目标，我们必须拥有 从来源于细胞裂解物的复杂起始材料获得高度纯化的蛋白质样品的能力， 重组蛋白表达系统。该提案要求资金取代一名14岁的（最近 已停产）AKTA FPLC蛋白质纯化仪器，具有来自同一台仪器的类似但最新版本 制造商所提出的系统，AKTA纯，是一个多功能和强大的液相色谱系统 用于蛋白质和蛋白质复合物的可靠纯化。它涵盖了广泛的色谱 该设备可满足各种应用，并满足提供最高纯度所需的自动化要求。仪器将 确保继续进行原裁决中提出的生物化学、生物物理和结构研究。

Abstract This request for a supplement to the grant R01GM135592 proposes the acquisition of a liquid chromatography protein purification system to support the research aims that focus on investigating the substrate targeting mechanisms of the F-box subunit FBXO31 of the SCF ubiquitin ligase in a novel DesCEND pathway. By identifying and characterizing substrates of FBXO31 at both a systems level and the molecular level, we proposed to elucidate the complementary structural determinants of the FBXO31–degron pair that confer substrate specificity and dictate protein recognition and fate. To achieve our goals, it is vital that we have the ability to obtain highly purified protein samples from complex starting materials derived from cell lysates and recombinant protein expression systems. This proposal requests funds to replace a 14-year old (and recently defunct) AKTA FPLC protein purification instrument with a similar but up-to-date version from the same manufacturer. The proposed system, the AKTA pure, is a versatile and powerful liquid chromatography system for the reliable purification of proteins and protein complexes. It covers a broad range of chromatography applications and meets the automation requirements needed to deliver the highest purity. The instrument will ensure the continued biochemical, biophysical and structural studies proposed in the parent award.